Figure 8C, D

A. THP-1 cells were infected by spinoculation with an MOI of 20 with either nonfluorescent TB40/E, UL88-STOP, or UL88-Rev, the latter two of which express GFP driven from the IE2 promoter or were left uninfected. GFP fluorescence was measured by flow cytometry at d5 post infection. B. Schematic representation of the experiment methodology for C and D. At day 3, 2.5 x 105 THP-1, of which ~ 2.5 x 104 were HCMV-infected, were transferred onto a monolayer of ~ 2.5 x 105 HDFs and virus spread was monitored every three days by confocal imaging up to day 15. C. Representative images of TB40/E-GFP and UL88-STOP from day 3 and day 7 are shown. D. The spread of infection was quantified as the area of fluorescence using the NIS element software. Graph shows fold change in area of fluorescence for each virus. E. MRC-5 cells were infected with TB40/E-mCh or UL88-STOP-mCh virus at MOI of 0.05. Cells were harvested for RNA isolation at 7 dpi. Antiviral gene expression was profiled using a set of 84 ISGs by qPCR. Results are plotted as volcano plot depicting the modulated genes as compared between TB40/E WT HCMV and UL88-STOP HCMV infection. F. ISGs known to be modulated in HCMV infection are shown with their relative fold change in mRNA levels (normalized to GAPDH) between TB40/E WT and UL88-STOP virus infection. The graph shows the genes that remained unchanged (green box), downregulated (red box), or upregulated (blue box), in TB40/E WT vs UL88-STOP HCMV-infected cells. Results are from biological triplicates.